Aminoglycoside-induced ototoxicity can cause permanent hearing impairment in approximately one third of patients receiving these antibiotics, sometimes limiting therapeutic dosing. D-methionine (D-met), which we patented, licensed, and are now using in other FDA approved clinical trials, protects against amikacin- and gentamicin-induced hearing loss without antimicrobial interference in animal studies thus far. The purpose of these proposed studies is to optimize D-met dosing protocols and obtain further proof of concept data for gentamicin and amikacin otoprotection, to determine if D-met also protects against tobramycin-induced ototoxicity, and to ensure that D-met does not interfere with the antimicrobial action of these aminoglycosides or other antibiotics commonly administered concomitantly in clinical care. The long-term goal of the studies is to enable us to conduct safe and effective FDA approved clinical trials of D-met as an otoprotective agent for aminoglycoside-induced ototoxicity and obtain FDA NDA approval, thus improving patient care. D-met would be the first agent approved for that purpose. The specific aims are: 1) to determine if fractionating D-met daily dosing improves protection against aminoglycoside ototoxicity by comparing once versus twice daily dosing for the same daily dose in the gentamicin guinea pig model;2) to optimize dosing for D-met's otoprotection against gentamicin- and amikacin-induced ototoxicity by obtaining D-met dose response curves;3) to determine if D-met can protect against tobramycin-induced ototoxicity by first refining a guinea pig model for tobramycin ototoxicity and then obtaining D-met dose response curves in tobramycin-ototoxicity guinea pig models;and 4) to ensure that D-met does not interfere with the antimicrobial action of gentamicin, amikacin, or tobramycin, in isolation or in conjunction with other concomitant antibiotics used in clinical care. The outcome measures for the first three specific aims will be auditory brainstem response threshold shifts and inner and outer hair cell counts. For the fourth specific aim, in vitro outcomes include the Minimum Inhibitory Concentration, Minimum Bactericidal Concentration, and post-antibiotic effect, and in vivo study outcomes include survival and bacterial counts. These studies could improve public health by developing a new drug to reduce aminoglycoside-induced hearing loss without reducing the antibiotics'effectiveness and possibly allowing higher dosing to better control infectious diseases.